Ion channels in mammalian systems have been, and currently are, the subject of intensive scientific investigation because of the importance and variety of their biochemical functions. Ion channels are now understood to be polypeptide or protein structures with a tertiary-quaternary structure forming interior pores embedded in cell membrane walls, that can control the flow of ionic currents in response to either electrical excitation (voltage gating) or the presence of neurotransmitter (ligand gating). Current work shows that there are familial relationships or similarities in peptide sequence between the different types of voltage gated channels, such as those from mammalian brain, cardiac and skeletal muscles, including sodium, calcium, and potassium channels. Familial relationships within the ligand-gated types, such as acetylcholine receptors (AChR) (both neuronal and muscle), glycine receptors, and gamma aminobutyric acid (GABA) receptors also exist. More distant similarities also exist between these ion channels and ion channels derived from certain non-mammalian systems, studies of which have been instructive because of the accessibility of the proteins to analysis or the unique properties which the proteins exhibit. Research undertaken heretofore has initially concentrated on identifying the existence and general character and function of the channels. More recently it has broadened out greatly into detailed studies of the properties and interactions of specific ion channels, notably in electrical excitability and synaptic transmission with respect to the brain, heart and other muscle. This very important work has led to the development of a number of useful pharmaceuticals, such as the widely used channel blockers used to treat hypertension.
The functions of ion channel proteins have largely been studied by reconstituting the proteins from natural sources or replicating them by cloning techniques, and positioning them in a lipid bilayer membrane. The lipid bilayer duplicates the cell membrane to separate the aqueous ionic environment from the cell interior both physically and electrically while providing a support for the ion channel. The macromolecule forming the protein channel, positioned across the supporting lipid bilayer, can be coupled into a sensitive "patch clamp" circuit by which ionic current flowing through the channels can be precisely measured. The test procedures have been developed to the point at which "single channel" recordings can be made, so that the characteristics of individual "authentic" (or "native") channels can be studied. Using these techniques, the characteristics of many different ion channels have been precisely characterized from authentic, reconstituted, cloned and analog versions.
An ion channel does not simply open or close in response to a stimulus, but when activated functions in pulsed fashion or in what may be termed a relaxation mode. That is, it varies between open and closed states which last for intervals typically in the millisecond range. Furthermore, each type of ion generally exhibits a characteristic pattern of selectivity to different ions, and has a single channel conductance of predetermined amplitude under standard conditions. While reconstituted and expressed channel proteins can be generated for most types of ion channels they are often difficult and expensive to obtain, or unduly delicate, unstable and sensitive. Their use often gives rise to ambiguity when one seeks to alter or refine the protein chemistry. Efforts at protein synthesis of ion channels have heretofore sought to demonstrate ionic activity, but have not provided that degree of mimetic fidelity to the authentic channels that would permit meaningful use of the synthesized versions or assurance as to how improvements might be made. Examples are to be found in Oiki et al, Proc. Natl. Acad. Sci. USA, Vol. 85, April 1988, pp. 2393-2397, directed to the voltage-sensitive sodium channel, in Lear et al, Science, Vol. 240, (27 May 1988) pp. 1177-1181, and in Jullien et al, Tetrahedron Lett. 29, (1988) pp. 3803-3806. The Lear et al synthesis coupled protected helices to a solid support, and the helices were then cleared from the support. Further the protein ion channels were not intended to be mimetic, but were simply intended to show ionic permeability, being formed from repetitive sequences of two residues only Leucine for the apolar face of a helix and Serine for the polar face. The fact that an ion channel effect of higher fidelity to the authentic could be obtained was previously known, as evidenced by the Oiki et al reference, supra. In addition, it should be noted that Mutter et al, in Tetrahedron, 44:771-785 (1988) suggest that synthetic proteins may be made useful by linking amphipathic .alpha.-helix and .beta.-sheet peptides to a tethering synthetic peptide template. This teaching does not, however, supply the deficiencies of the synthesized ion channel structures so as to render them mimetic of the authentic model.
If the kinetics and sensitivity of a synthetic ion channel do not correspond to the authentic channel, in an adequately mimetic way, then the synthetic channel is not useful to screen physiologically active substrates in a way useful to predict activity in human subjects. Non-mimetic proteins can be used only as a basis for further studies in molecular mechanics. To analyze a particular channel function in a meaningful way, or to screen chemicals to ascertain the precise nature of their pharmacological properties, a number of family-related synthetic ion channels are needed.
To achieve such results, however, major barriers must be overcome. The macromolecular structure of the sensing protein or transport site a channel protein cannot be exactly elucidated, as yet, using modern analytical instrumentation. The molecular structures are complex, and the exterior and interior geometries and encoding patterns which give rise to functional properties have not heretofore been fully understood and have not been predictable. Since the crucial functional attributes can only be measured indirectly, in electrical terms, the molecular dynamics are only partially understood or theoretically analyzed. The authentic voltage gated ion channel is believed to have an interior pore or aligning structure which enables ionic flow to take place, but that it also incorporates a sensor which is responsive to the triggering event and controls gating by some type of conformational change. The channels define a central-waterlike transmembrane pore or pathway of cylindrical shape with a converging lumen near the end. Internally, the macromolecule is regarded as having multiple subunits comprising .alpha.-helices of characteristic types. The active or gating portion of the channel is believed to comprise a chemically selective sensor for the ligand gated structure or electrically active adjacent helices forming ion pairs within the voltage gated structures. The ion channel must confine an aqueous solution within the cell, must be fixed into the lipid membrane or onto a hydrolyzable peptide at its outer side, as well as insulate the active interior portions form the membrane. The pore cross-sectional dimension and cross-sectional area must also be compatible with the authentic channel. Further the synthesized peptides must fold into an energy minimized structure and in proper orientation to the membrane with hydrophilic and lipophilic sides in proper relation. Thus a substantial need exists for a synthetic protocol that can provide a single accurately mimetic synthetic channel protein. Further, the need to provide a generally applicable methodology to produce families of mimetics which is an even more complex problem remains unfulfilled.
The signal transducing properties of ion channels, their ability to provide net signal gain, their high sensitivity and their minimal size are a few of the factors which have led some to contemplate their use as biosensor. Examples are found in U.S. Pat. Nos. 4,637,861; 4,661,235; 4,776,944; 4,824,529 and 4,849,343, and also in Statutory Invention Registration H201. The ionic current characteristics of an ion channel are not functional unless it is embedded in, and provides a conductive pathway through, a dielectric or very high resistance (of the order of 10.sup.15 ohms) membrane, typically a lipid bilayer. Nonetheless the dominant and indispensable portion of this combination is the dynamic, event-responsive, ion channel. The referenced patents are however directed to the much simpler objective of stabilizing and strengthening a membrane. The production of active biosensor in the form of the instability, fragility and complexity of ion channels, rather than the characteristics of the membrane, is what has blocked the development of active biosensor of this class.